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Environmental Compliance and Control for Radiopharmaceutical Production
Published in Michael Ljungberg, Handbook of Nuclear Medicine and Molecular Imaging for Physicists, 2022
Ching-Hung Chiu, Ya-Yao Huang, Wen-Yi Chang, Jacek Koziorowski
Bacterial endotoxin is the lipopolysaccharide (LPS) component of the cell wall of gram-negative bacteria. It is pyrogenic, and it is a risk to patients who are administered intravenous and intramuscular preparations [21]. The pathological effects of endotoxin, when injected, are a rapid increase in body temperature followed by extremely rapid and severe shock, often followed by death, before the cause is even diagnosed. The limulus amebocyte lysate (LAL) test is the most widely method used for endotoxin tests [22]. The pharmacopoeia monographs for the LAL test are long-established and relatively comprehensive and have been applied to the testing of parenteral products for bacterial endotoxin since the 1980s. Pyrogens are a concern for pharmaceutical drug products and for many of the ingredients used to formulate them. This is especially for radiopharmaceuticals that have direct contact with human blood. Here, by far the most concerning pyrogen is bacterial endotoxin. In relation to this, the risks of endotoxin to radiopharmaceutical processing and some of the control measures in place to reduce the risk of endotoxin contamination should be considered.
Breast Imaging with Positron Emission Tomography
Published in Raymond Taillefer, Iraj Khalkhali, Alan D. Waxman, Hans J. Biersack, Radionuclide Imaging of the Breast, 2021
Hans Bender, Holger Palmedo, Hans J. Biersack, Axel Schomburg
Total volume and radioactive concentration. Radionuclide purity: gamma ray spectroscopy using a sodium iodide detector and a multichannel analyzer.Radiochemical purity: high-pressure liquid chromatography (HPLC) against an FDG standard using refractive index and gamma ray detectors. Chemical purity: thin-layer chromatography against a Kryptofix 222 standard.Radionuclide identity: decay counting of the final product over a 10-min period in a dose calibrator to verify the radionuclide half-life.pH: narrow range pH strip and compared to pH=6 and pH=7 buffers.Sterility: incubation of final product in culture media; the medium is inspected for bacterial growth during a 14-day incubation at 30 to 35°C and 20 to 25°C. Due to the short half-life, sterility is obtained by sterile filtration (0.22-μm filter). Control samples can be stored at -20/70°C, which allows sterility testing in doubtful cases, any time later. Bacterial endotoxins: limulus amebocyte lysate (LAL) against standard endotoxin.
Endotoxin Detection in Body Fluids: Chemical Versus Bioassay Methodology
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
In the context of this review, chemical assays for endotoxin are those defined as analytical procedures that recognize and quantify either the entire endotoxin molecule or any part of the endotoxin molecule through a chemical reaction or signal, including binding to a specific ligand. It can be argued that the Limulus amebocyte lysate test, which relies on the activation of the clotting cascade isolated from the blood of the horseshoe crab and has been shown to correlate quite well with a number of physiological responses to endotoxin, is a bioassay. LAL will, however, be treated here as a chemical test, since endotoxin reacts with an LAL enzyme (factor C) and does not require a living cell or cellular system in order to elicit a dose-dependent response.
Structure-based engineering of a novel CD3ε-targeting antibody for reduced polyreactivity
Published in mAbs, 2023
Catherine Y Liu, Cory L Ahonen, Michael E Brown, Ling Zhou, Martin Welin, Eric M Krauland, Robert Pejchal, Paul F Widboom, Michael B Battles
IgG-like (1 + 1) bispecific antibodies were produced in CHO-K1 cells after transient transfection with four expression vectors encoding a CD20 or HEL antigen-binding heavy chain, a CD20 or HEL antigen-binding light chain, a CD3-binding heavy chain, and a CD3-binding light chain. HC/LC pairing and CH3 heterodimerization mutations were introduced to facilitate recovery of heterodimers (details disclosed in the patent literature).75,76 Bispecific antibody molecules were harvested from culture supernatants and purified by ProA affinity chromatography followed by Mono S cation exchange polishing using a linear pH gradient as previously described.77 Purified bispecifics were characterized by liquid chromatography-mass spectrometry (LC/MS), analytical SEC, and analytical IEX. Bispecific molecules purified by ProA and subsequent cation exchange as described above, were shown to contain less than 1% homodimer and less than 1% aggregates. Endotoxin levels were measured using a limulus amebocyte lysate assay (Charles River Laboratories) and were shown to be below 0.1 EU/mg.
Interaction of nanoparticles with endotoxin Importance in nanosafety testing and exploitation for endotoxin binding
Published in Nanotoxicology, 2021
Maria Mangini, Alessandro Verde, Diana Boraschi, Victor F. Puntes, Paola Italiani, Anna Chiara De Luca
The LAL test is based on the use of lysates from amoebocytes (blood cells) of the horseshoe crab Limulus polyphemus. Exposure to LPS induces a defensive reaction in the cells, which includes the LPS-dependent activation of Factor C, an enzyme that initiates a coagulation/clotting cascade aiming to inhibit the infection (Levin and Bang 1964a, 1964b). Clotting can also be induced by β-(1,3)-d-glucan, a molecule present in fungal pathogens, which activates another clotting enzyme, Factor G, which initiates the same defensive coagulation cascade (Morita et al. 1981). The LAL assay exploits the high reactivity of the Factor C present in the lysate of Limulus amoebocytes for an accurate quantitative detection of the LPS biological activity. Several LAL assays have been developed and are available commercially, from the original clotting and turbidimetric assays to the more sensitive assays that use synthetic chromogenic or otherwise tagged substrates. In these assays, LPS-activated Factor C induces the release of a colored/fluorescent/luminescent molecule from the synthetic substrate, which can be precisely measured. The sensitivity of the LAL tests ranges from 0.005 to 50 EU/mL depending on the sample source, bacterial species, strain, and detection method. To avoid the false positives due to the possible presence of β-glucans, many LAL assay kits available on the market include reagents that specifically block the Factor G-dependent reaction.
Cytotoxicity profiles of multi-walled carbon nanotubes with different physico-chemical properties
Published in Toxicology Mechanisms and Methods, 2020
Katsuhide Fujita, Sawae Obara, Junko Maru, Shigehisa Endoh
The concentration of MWCNTs in the six stock suspensions (MW1–4: 1.0 mg/mL; MW5 and 6: 0.5 mg/mL) was determined by measuring their ultraviolet (UV)-visible absorption spectra using a UV-2550 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at wavelengths of 600–800 nm (Fujita et al. 2017). The stock suspensions were adjusted to a 10- to 1000-fold dilution series to the required concentration in cell culture medium for use in in vitro cell-based assays and then used for a battery of genotoxicity tests. The zeta potentials of MWCNTs dispersed in the stock suspensions were measured using a Zetasizer Nano zeta potential analyzer (Malvern Panalytical Ltd., Malvern, UK). Hitachi H-7000 and H-7100 transmission electron microscopy (TEM) systems at 75 kV and 100 kV (Hitachi, Ltd., Tokyo, Japan), respectively, were used to observe MWCNTs in the stock suspensions. The lengths and fiber diameters of the MWCNTs were measured from ∼1000 or 200 MWCNTs bundles using a JEOL JEM-1010 TEM (Jeol Ltd., Tokyo, Japan) at 100 kV. A Limulus amebocyte lysate test (Associates of Cape Cod, Inc., East Falmouth,MA, USA; data not shown) was conducted to find endotoxins in the six MWCNT stock suspensions, but none was detected.